PROJECT SUMMARY A majority of neurons live throughout the life of an organism. While extrinsic survival cues regulating neuronal cell death are well known, intrinsic mechanisms contributing to neuronal survival are under study. We have carried out a systematic investigation of whether and how neuronal vulnerability to apoptosis is intrinsically controlled to favor longevity. Our preliminary results show that increasing the threshold to apoptosis is initiated as early as the onset of neurogenesis and is accompanied by neuronal maturation. Unbiased proteomic and transcriptomic searches for underlying molecular attributes identify a couple of pro-apoptotic genes including Bak1. BAK1 protein is completely turned off in adult neural tissues. We found that tissue-specific expression of BAK1 is controlled at the RNA level by Alternative Splicing coupled Nonsense-mediated mRNA Decay (AS- NMD). This is reminiscent of the regulation of tissue-specific expression of Psd-95, also by AS-NMD. Based on these premises, we hypothesize that AS-NMD is commonly used to enforce neural-specific gene expression and the AS-NMD repression of Bak1 programs cells? intrinsic sensitivity to apoptosis during neuronal differentiation. We will apply new and robust experimental strategies to determine the true breadth of AS-NMD regulation in the brain. We will systematically determine the trans- and cis-regulation of AS-NMD-targeted pro- apoptotic genes and reveal their impacts on determining neurons? increased resistance to apoptosis. Our long history of researching neuronal survival, alternative splicing, and NMD in the brain places us in a unique position to advance these fields. Our team, with complementary expertise in genetics, neurobiology, molecular cellular biochemical, and computational biology, has demonstrated successful collaborations. The completion of this project will establish AS-NMD as a new regulatory mechanism of tissue-specific gene expression independent of transcriptional and miRNA-mediated post-transcriptional controls. The results of the proposed research will also unveil a novel regulatory mechanism for apoptosis and showcase the new functional significance of alternative splicing and NMD regulation, which have far-reaching impacts in development and disease.